Pd-l1-specific antibodies and methods of using the same

ABSTRACT

The present disclosure relates generally to antibodies and functional binding fragments thereof that bind to programmed death-ligand 1 (PD-L1). In particular, the disclosed antibodies and fragments bind to human PD-L1 and comprise novel complementary determining regions (CDRs) also disclosed herein. Finally, the present disclosure relates to administering the disclosed antibodies and fragments to subjects with cancer, thereby treating or slowing the progression or proliferation of the cancer.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 19, 2017, isnamed 099263-0801_SL.txt and is 43,085 bytes in size.

FIELD OF INVENTION

The present disclosure relates generally to antibodies and functionalbinding fragments thereof that bind to programmed death-ligand 1(PD-L1). In particular, the disclosed antibodies and fragments bind tohuman PD-L1 and comprise novel complementary determining regions (CDRs).Finally, the present disclosure relates to administering the disclosedantibodies and fragments to subjects with cancer, thereby treating orslowing the progression or proliferation of the cancer.

BACKGROUND

PD-L1 (formerly B7-H1) is a B7 family member that is expressed on manycell types, including antigen-presenting cells (“APCs”) and activated Tcells (Yamazaki et al. (2002) J. Immunol. 169:5538). PD-L1 binds to bothPD-1 (CD279) and B7-1. Both binding of T-cell-expressed B7-1 by PD-L1and binding of T-cell-expressed PD-L1 by B7-1 result in T cellinhibition (Butte et al. (2007) Immunity 27:111). There is also evidencethat, like other B7 family members, PD-L1 can also provide costimulatorysignals to T cells (Subudhi et al. (2004) J. Clin. Invest. 113:694;Tamura et al. (2001) Blood 97:1809). Furthermore, expression of PD-L1 onthe cell surface has also been shown to be upregulated through IFN-γstimulation.

The interaction between PD-1 and its ligand partners PD-L1 (B7-H1;CD274) and PD-L2 (B7-DC; CD273) is an important negative costimulatorysignaling pathway involved in the regulation of T cell activation. PD-1can be expressed on T cells, B cells, natural killer T cells, activatedmonocytes and dendritic cells (DCs). PD-1 is expressed by activated, butnot by unstimulated, human CD4⁺ and CD8⁺ T cells, B cells and myeloidcells. Nishimura et al., Int. Immunol. 8: 773-80 (1996); Boettler etal., J. Virol. 80: 3532-40 (2006). There are at least 4 variants of PD-1that have been cloned from activated human T cells, includingtranscripts lacking (i) exon 2, (ii) exon 3. (iii) exons 2 and 3 or (iv)exons 2 through 4. Nielsen et al., Cell. Immunol. 235: 109-16 (2005).

Normal human tissues seldom express PD-L1 protein on their cell surface,with the exception of tonsil, placenta, and a small fraction ofmacrophage-like cells in lung and liver, suggesting that under normalphysiological conditions, PD-L1 mRNA is under tight posttranscriptionalregulation. In sharp contrast, PD-L1 protein is abundantly expressed onthe cell surface in various human cancers, as indicated by Chen & Han,Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future. J.CLIN. INVEST. 125, 3384-3391 (2015).

PD-L2 expression, on the other hand, is more restricted than PD-L1. Forinstance, PD-L2 is inducibly expressed on DCs, macrophages, and bonemarrow-derived mast cells.

Additionally, several studies show a receptor for PD-L1 that isindependent of PD-1. B7.1 has also been identified as a binding partnerfor PD-L1. Butte et al., Immunity 27: 111-22 (2007). Chemicalcrosslinking studies suggest that PD-L1 and B7.1 can interact throughtheir IgV-like domains. B7.1:PD-L1 interactions can induce an inhibitorysignal into T cells. Ligation of PD-L1 on CD4+ T cells by B7.1 orligation of B7.1 on CD4+ T cells by PD-L1 delivers an inhibitory signal.T cells lacking CD28 and CTLA-4 show decreased proliferation andcytokine production when stimulated by anti-CD3 plus B7.1 coated beads.In T cells lacking all the receptors for B7.1 (i.e., CD28, CTLA-4 andPD-L1), T cell proliferation and cytokine production were no longerinhibited by anti-CD3 plus B7.1 coated beads. This indicates that B7.1acts specifically through PD-L1 on the T-cell in the absence of CD28 andCTLA-4. Similarly, T cells lacking PD-1 showed decreased proliferationand cytokine production when stimulated in the presence of anti-CD3 plusPD-L1 coated beads, demonstrating the inhibitory effect of PD-L1ligation on B7.1 on T cells. When T cells lacking all known receptorsfor PD-L1 (i.e., no PD-1 and B7.1), T cell proliferation was no longerimpaired by anti-CD3 plus PD-L1 coated beads. Thus, PD-L1 can exert aninhibitory effect on T cells either through B7.1 or PD-1.

In the context of human disease, PD-L1 expression has been found inseveral murine and human cancers, including human lung, ovarian andcolon carcinoma and various myelomas (Iwai et al. (2002) PNAS99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53). PD-L1has been suggested to play a role in tumor immunity by increasingapoptosis of antigen-specific T-cell clones (Dong et al. (2002) Nat Med8:793-800). It has also been suggested that PD-L1 might be involved inintestinal mucosal inflammation and inhibition of PD-L1 suppresseswasting disease associated with colitis (Kanai et al. (2003) J Immunol171:4156-63).

As a result, therapeutic targeting of PD-L1 is an area of intensemedical interest. The inhibition of PD-L1 signaling has been proposed asa means to enhance T cell immunity for the treatment of cancer (e.g.,tumor immunity) and infection, including both acute and chronic (e.g.,persistent) infection. However, because only one targeted anti-PD-L1therapeutic has received marketing approval from the FDA to date, asignificant unmet medical need exists.

SUMMARY

The present disclosure provides isolated antibodies, in particular humanantibodies that bind to PD-L1 and exhibit desirable therapeuticproperties. These properties include high affinity binding to PD-L1,specifically human PD-L1.

In one aspect, the disclosure relates to antibodies or functionalfragments thereof that bind to human programmed death-ligand 1 (PD-L1),wherein the antibody or functional fragment thereof comprises: a heavychain comprising: CDRH1 comprising amino acids 1-3 and 7-9 of SEQ ID NO:1, wherein amino acids 4-6 are not SSY; CDRH2 comprising amino acids9-17 of SEQ ID NO: 8; and CDRH3 comprising SEQ ID NO: 15; and a lightchain comprising CDRL1 comprising SEQ ID NO: 20; CDRL2 comprising SEQ IDNO: 21; and CDRL3 comprising SEQ ID NO: 22, SEQ ID NO: 75, or SEQ ID NO:76.

In some embodiments, the CDRH1 region comprises an amino acid sequenceselected from the group consisting of SEQ ID NOS: 2, 3, 4, 5, 6, and 7,and in some embodiments the CDRH2 region comprises an amino acidsequence selected from the group consisting of SEQ ID NOS: 8, 9, 10, 11,12, 13 and 14.

In some embodiments, the variable heavy framework region 1 comprises SEQID NO. 26 or SEQ ID NO: 27. In some embodiments, the variable heavyframework region 2 comprises SEQ ID NO. 28, the variable heavy frameworkregion 3 comprises SEQ ID NO. 29, and/or the variable heavy frameworkregion 4 comprises SEQ ID NO. 30.

In some embodiments, the variable light framework region 1 comprises SEQID NO. 31, the variable light framework region 2 comprises SEQ ID NO.32, the variable light framework region 3 comprises SEQ ID NO. 33, SEQID NO. 77, or SEQ ID NO. 78, and/or the variable light framework region4 comprises SEQ ID NO. 34.

In some embodiments, the variable heavy chain sequence comprises anamino acid sequence selected from the group consisting of SEQ ID NOS:36, 37, 38, 39, 40 and 41. In other embodiments, the variable heavychain sequence comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 51, 52, 53, 54, 55, and 56.

In some embodiments, the variable light chain sequence comprises SEQ IDNO: 42.

In some embodiments, the antibody or functional fragment thereofexhibits antibody dependent cellular cytotoxic (ADCC) activity, theantibody or functional fragment thereof exhibits antitumor activity, theantibody or functional fragment thereof inhibits the binding of PD-L1 toPD-1, or the antibody or functional fragment thereof prevents PD-1mediated inhibition of T-cell activation.

In some embodiments, the antibody or functional fragment thereof is alow fucose antibody or functional fragment thereof, and in someembodiments, the antibody or functional fragment thereof isdefucosylated or afucosylated.

In another aspect, the disclosure relates to antibodies or functionalfragments thereof that binds to human programmed death-ligand 1 (PD-L1),wherein the antibody or functional fragment thereof comprises a heavychain comprising SEQ ID NO: 35.

In another aspect, the disclosure relates to antibodies or functionalfragments thereof that bind to human programmed death-ligand 1 (PD-L1),wherein the antibody or functional fragment thereof comprises: a heavychain comprising: CDRH1 comprising SEQ ID NO: 16; CDRH2 comprising SEQID NO: 17; and CDRH3 comprising amino acids 6-13 of SEQ ID NO: 18; and alight chain comprising: CDRL1 comprising SEQ ID NO: 23; CDRL2 comprisingSEQ ID NO: 24; and CDRL3 comprising SEQ ID NO: 25.

In some embodiments, the CDRH3 comprises SEQ ID NO: 18, and in someembodiments the CDRH3 comprises SEQ ID NO: 19.

In some embodiments, the variable heavy framework region 1 comprises SEQID NO. 49, the variable heavy framework region 2 comprises SEQ ID NO.28, the variable heavy framework region 3 comprises SEQ ID NO. 50,and/or the variable heavy framework region 4 comprises SEQ ID NO. 30.

In some embodiments, the variable light framework region 1 comprises SEQID NO. 45, the variable light framework region 2 comprises SEQ ID NO.46, the variable light framework region 3 comprises SEQ ID NO. 47,and/or the variable light framework region 4 comprises SEQ ID NO. 34.

In some embodiments, the variable heavy chain sequence comprises SEQ IDNO: 48 or 49, and in some embodiments, the variable light chain sequencecomprises SEQ ID NO: 50.

In some embodiments, the antibody or functional fragment thereofexhibits antibody dependent cellular cytotoxic (ADCC) activity, theantibody or functional fragment thereof exhibits antitumor activity, theantibody or functional fragment thereof inhibits the binding of PD-L1 toPD-1, or the antibody or functional fragment thereof prevents PD-1mediated inhibition of T-cell activation.

In some embodiments, the antibody or functional fragment thereof is alow fucose antibody or functional fragment thereof, and in someembodiments, the antibody or functional fragment thereof isdefucosylated or afucosylated.

Some embodiments relate to a pharmaceutical composition comprising thedisclosed antibodies or functional fragments thereof.

In another aspect, the disclosure relates to methods of inhibitingcancer cell proliferation in a subject, comprising administering thedisclosed antibodies or functional fragments thereof to a subject withcancer. For instance, in some embodiments, the disclosure relates tomethods of inhibiting tumor cell proliferation in a subject, comprisingadministering an antibody or functional fragment thereof that bindshuman PD-L1 with a KD of at least 2.50×10-8 and CDRH3 comprising aminoacids 6-13 of SEQ ID NO: 18 to a subject with cancer. Likewise, in someembodiments, the disclosure relates to methods of inhibiting cancer cellproliferation in a subject, comprising administering an antibody orfunctional fragment that binds human PD-L1 with a KD of at least8.30×10-9 and CDRH3 comprising SEQ ID NO: 15 to a subject with cancer.

In some embodiments, the cancer is a hematological cancer, aneurological cancer, breast cancer, a gastrointestinal cancer, renalcell carcinoma, or a genitourinary cancer. In particular, in someembodiments, the gastrointestinal cancer is colon cancer, thegenitourinary cancer is ovarian cancer, prostate cancer or bladdercancer; the hematological cancer is lymphoma. Non-Hodgkin's lymphoma,chronic lymphocytic leukemia, or multiple myeloma; or the renal cellcarcinoma is clear cell renal cell carcinoma.

In some embodiments, the cancer is melanoma, lung cancer (such asnon-small cell lung cancer), head and neck cancer, liver cancer,pancreatic cancer, bone cancer, or a vascular cancer.

In some embodiments, the cancer is a highly immunogenic carcinoma or thecancer is a PD-L1 expressing cancer.

In some embodiments, the methods further comprise a step ofadministering to the subject an additional therapeutic compound. Forexample, in some embodiments, the additional therapeutic compound is a Tcell expressing a chimeric antigen receptor (CAR), a tumor-targetingantibody, an immune response potentiating modality, or a small moleculedrug.

In some embodiments, the immune response potentiating modality is ananti-GITR antibody, an anti-OX40 antibody, an anti-CD137 antibody, or aTLR agonist.

In some embodiments, the tumor-targeting antibody is an anti-CAIXantibody.

In some embodiments, the small molecule drug is a tumor-targeting smallmolecule drug, such as a BTK inhibitor, an EGFR inhibitor, a PARPinhibitor, a BET inhibitor, a BRAF inhibitor, or a PI3Kdelta inhibitor.

The foregoing general description and following detailed description areexemplary and explanatory and are intended to provide furtherexplanation of the disclosure as claimed. Other objects, advantages, andnovel features will be readily apparent to those skilled in the art fromthe following brief description of the drawings and detailed descriptionof the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the percent inhibition of PD-1 binding to PD-L-1+ cells byanti-PD-L1 antibodies. The results were obtained using FACS analysis.

FIG. 2 shows the binding kinetics of exemplary anti-PD-L1 antibodyCTI-48 against (A) human PD-L, (B) mouse PD-L1, and (C) cyno PD-L1.

FIG. 3 shows that exemplary anti-PD-L1 antibody CTI-48 exhibits ADCCactivity on PD-L+ lymphoma cells with primary NK cells.

FIG. 4 shows reversal of T-cell inhibition with PD-L1 in a reporter(NFAT) bioassay of immunoblockade with select anti-PD-L1 antibodies.

FIG. 5 shows blocking of PD-L1 binding to B7.1 by CTI-48 and a clinicalcontrol mAb.

FIG. 6 shows the effect of the disclosed antibodies on IFN-γ production.Antibodies were dosed into mixed lymphocyte reaction (MLR) cultures at aconcentration of 10 μg/mL. Data were normalized to vehicle control andare presented as combined mean+SEM (n=6). *p<0.05 **p<0.01, ***p<0.001indicates statistical significance when compared to appropriate isotypecontrol (hIgG1) using Ordinary one-way ANOVA with Dunnett's multiplecomparison post-hoc test. This figure shows a side-by-side comparison ofCTI-48 and a clinical control mAb.

DETAILED DESCRIPTION

The compositions and methods of the present disclosure employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, MolecularCloning: A Laboratory Manual, second edition (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture(R. I. Freshney, ed., 1987); Methods in Enzymology (Academic Press,Inc.); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds1987, and periodic updates); PCR: The Polymerase Chain Reaction, (Mulliset al., ed., 1994); A Practical Guide to Molecular Cloning (PerbalBernard V., 1988); Phage Display: A Laboratory Manual (Barbas et al.,2001).

I. Definitions

It is to be understood that methods are not limited to the particularembodiments described, and as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting. Thescope of the present technology will be limited only by the appendedclaims.

As used herein, certain terms may have the following defined meanings.As used in the specification and claims, the singular form “a,” “an” and“the” include singular and plural references unless the context clearlydictates otherwise. For example, the term “a cell” includes a singlecell as well as a plurality of cells, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the composition or method. “Consisting of” shall meanexcluding more than trace elements of other ingredients for claimedcompositions and substantial method steps. Embodiments defined by eachof these transition terms are within the scope of this disclosure.Accordingly, it is intended that the methods and compositions caninclude additional steps and components (comprising) or alternativelyincluding steps and compositions of no significance (consistingessentially of) or alternatively, intending only the stated method stepsor compositions (consisting of).

As used herein, “about” means plus or minus 10%.

As used herein, “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances where it does not.

As used herein, the terms “individual”, “patient”, or “subject” can bean individual organism, a vertebrate, a mammal (e.g., a bovine, acanine, a feline, or an equine), or a human. In a preferred embodiment,the individual, patient, or subject is a human.

As used herein, the term an “isolated antibody” is intended to refer toan antibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to PD-L1 is substantially free of antibodies that donot bind to PD-L1). An isolated antibody that specifically binds to anepitope of PD-L1 may, however, have cross-reactivity to other proteins,such as PD-L2 or PD-1, as well as proteins from different species.However, the antibody preferably always binds to human PD-L1. Inaddition, an isolated antibody is typically substantially free of othercellular material and/or chemicals.

As used herein, the term “humanized antibody” refers to an antibody thatcomprises the CDRs of antibodies derived from mammals other than human,and the framework (FR) region and the constant region of a humanantibody. A humanized antibody is useful as an effective component in atherapeutic agent according to the present disclosure since antigenicityof the humanized antibody in human body is lowered.

As used herein, the term “recombinant human antibody” includes all humanantibodies that are prepared, expressed, created or isolated byrecombinant means, including but not limited to (a) antibodies isolatedfrom an animal (e.g., a mouse) that is transgenic or transchromosomalfor human immunoglobulin genes or a hybridoma prepared therefrom, (b)antibodies isolated from a host cell transformed to express the antibody(e.g., from a transfectoma), (c) antibodies isolated from a recombinant,combinatorial human antibody library, and (d) antibodies prepared,expressed, created or isolated by any other means that involve splicingof human immunoglobulin gene sequences to other DNA sequences. Suchrecombinant human antibodies have variable and constant regions derivedfrom human germline and/or non-germline immunoglobulin sequences. Incertain embodiments, however, such recombinant human antibodies can besubjected to in vitro mutagenesis (or, when an animal transgenic forhuman Ig sequences is used, in vivo somatic mutagenesis) and thus theamino acid sequences of the VH and VL regions of the recombinantantibodies are sequences that, while derived from and related to humangermline VH and VL sequences, may not naturally exist within the humanantibody germline repertoire in vivo.

As used herein, the term “glycosylation pattern” is defined as thepattern of carbohydrate units that are covalently attached to a protein,more specifically to an immunoglobulin protein.

As used herein, the phrases “therapeutically effective amount” and“therapeutic level” mean that drug dosage or plasma concentration in asubject, respectively, that provides the specific pharmacological effectfor which the drug is administered in a subject in need of suchtreatment, i.e. to reduce, ameliorate, or eliminate the symptoms oreffects of cancer, malignant disease, or cancer cell proliferation. Itis emphasized that a therapeutically effective amount or therapeuticlevel of a drug will not always be effective in treating theconditions/diseases described herein, even though such dosage is deemedto be a therapeutically effective amount by those of skill in the art.The therapeutically effective amount may vary based on the route ofadministration and dosage form, the age and weight of the subject,and/or the subject's condition, including the type and stage of thecancer, malignant disease, or cancer cell proliferation, among otherfactors.

The term “highly immunogenic cancer” refers to cancers that have beeninfiltrated with T cells or lymphocytes or have developed a similarlymphatic response. In many instances, tumor immunogenicity is believedto be associated with increased rate of mutations. In this way, the moremutations a tumor has, the higher the chance that the tumor antigens cantrigger an immune response.

The terms “treatment” or “treating” as used herein with reference tocancer, malignant disease, or cancer cell proliferation refer toreducing, ameliorating or eliminating one or more symptoms or effects ofcancer, malignant disease, or cancer cell proliferation.

II. Anti-PD-L1 Antibodies

Provided herein are anti-PD-L1 antibodies that may be used, among otherreasons, to treat cancer. The anti-PD-L1 antibodies of the presentdisclosure are believed to enhance co-stimulation of the host immuneresponse through antagonism of at least one negative costimulatorysignal attributable to PD-L1.

The disclosed anti-PD-L1 antibodies and functional fragments thereofwill have a variety of functional properties for treating cancers ormalignant disease, including but not limited to having antibodydependent cellular cytotoxic (ADCC) activity, having antitumor activity,inhibiting the binding of PD-L1 to PD-1, and preventing PD-1 mediatedinhibition of T-cell activation.

Further, as a result of the antagonism of signaling through PD-L1,including blocking PD-L1 from interacting with either PD-1, B7.1 orboth, the disclosed antibodies and functional fragments will preventPD-L1 from sending a negative costimulatory signal to T-cells and otherantigen presenting cells, thus enhancing anti-tumor immunity and theimmunological defense against cancer and malignant disease. In addition,the disclosed anti-PD-L1 antibodies and functional fragments, may becombined with additional therapeutic compounds, including but notlimited to, CAR T cells (e.g., modified T cells that express ananti-CD19, anti-CAIX, anti-IL13Ra2, or anti-PD-L1 CAR), othertumor-targeting antibodies (e.g., an anti-CAIX antibody), immuneresponse potentiating modalities (e.g., an anti-GITR antibody, ananti-OX40 antibody, an anti-CD137 antibody, or a TLR agonist), and smallmolecule drugs (e.g., a BTK inhibitor, an EGFR inhibitor, a BETinhibitor, a PI3Kdelta inhibitor, a BRAF inhibitor, or a PARPinhibitor).

The disclosed antibodies can be polyclonal, monoclonal, chimeric, human,partially or fully humanized, and/or recombinant. For example, in someembodiments, the anti-PD-L1 antibody is a polyclonal antibody or aPD-L1-binding functional fragment thereof. In some embodiments, theanti-PD-L1 antibody is a monoclonal antibody or a PD-L1-bindingfunctional fragment thereof. In some embodiments, the antibodies andfunctional fragments thereof can bind human, cyno, and/or murine PD-L1.

Polyclonal antibodies may be obtained by methods known in the art, suchas by immunizing a selected animal with a PD-L1 antigen, collectingserum from the animal, and isolating and/or purifying antibodies fromthe serum. Monoclonal antibodies (mAbs) may be obtained by methods knownin the art, for example, by fusing antibody-producing cells withimmortalized cells to obtain a hybridoma, and/or by generating mAbs frommRNA extracted from bone marrow and spleen cells of immunized animalsusing combinatorial antibody library technology. Recombinant antibodiesmay be obtained by methods known in the art, for example, using phage oryeast display technologies and/or expressing or co-expressing antibodypolypeptides. Other techniques for making antibodies are known in theart, and can be used to obtain antibodies used in the methods describedherein.

Typically, an antibody consists of four polypeptides: two identicalcopies of a heavy (H) chain polypeptide and two copies of a light (L)chain polypeptide. Typically, each heavy chain contains one N-terminalvariable (VH) region and three C-terminal constant (CH1, CH2 and CH3)regions, and each light chain contains one N-terminal variable (VL)region and one C-terminal constant (CL) region. The variable regions ofeach pair of light and heavy chains form the antigen binding site of anantibody.

The terms “PD-L1-binding functional fragment” or “functional fragment,”as used herein, refer to one or more fragments of an anti-PD-L1 antibodythat retain the ability to bind PD-L1. Examples of binding fragmentsinclude (i) Fab fragments (monovalent fragments consisting of the VL,VH, CL and CH1 domains); (ii) F(ab′)2 fragments (bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion); (iii) Fd fragments (comprising the VH and CH1 domains); (iv) Fvfragments (comprising the VL and VH domains of a single arm of anantibody), (v) dAb fragments (comprising a VH domain); and (vi) isolatedcomplementarity determining regions (CDR), e.g., VH CDR3. Other examplesinclude single chain Fv (scFv) constructs. See e.g., Bird et al.,Science, 242:423-26 (1988); Huston et al., Proc. Natl. Acad. Sci. USA,85:5879-83 (1988). Other examples include PD-L1-binding-domainimmunoglobulin fusion proteins comprising (i) a PD-L1-binding domainpolypeptide (such as a heavy chain variable region, a light chainvariable region, or a heavy chain variable region fused to a light chainvariable region via a linker peptide) fused to an immunoglobulin hingeregion polypeptide, (ii) an immunoglobulin heavy chain CH2 constantregion fused to the hinge region, and (iii) an immunoglobulin heavychain CH3 constant region fused to the CH2 constant region.

The hinge region of the disclosed antibodies may be modified byreplacing one or more cysteine residues with, for example, serineresidues, to prevent dimerization. See, e.g., U.S. Patent ApplicationPublication 2003/0118592; U.S. Patent Application Publication U.S.2003/0133939. Additionally, in some embodiments, the disclosedantibodies may comprise other mutations, including but not limited to avariant Fc portion of an IgG having the point mutations S239D/I332E,S239D, or 1332E, or any combination thereof, or a variant Fc portion ofan IgG4 having the point mutation S228P. Such modifications may alterthe binding of the disclosed antibodies and functional fragments to Fcreceptors (FcRs), and in some embodiments, the antibody may be modifiedto be more stable, while in some embodiments, the antibody may bemodified to enhance ADCC function. When determining the number of theresidue, the Kabat numbering of residues may be determined for a givenantibody by alignment at regions of homology of the sequence of theantibody with a “standard” Kabat numbered sequence.

In some embodiments, the glycosylation patterns of the disclosedantibodies may be modified or altered. For instance, in someembodiments, the disclosed antibodies and functional fragments thereofmay be low fucose antibodies or they may be defucosylated or theantibodies may be expressed or produced in such a way that they arelacking fucose altogether (i.e. afucosylated). Modifying the fucosecontent of the antibody or functional fragment may be accomplishedthrough various means known in the art, for instance, expressing theantibody or functional fragment in a cell that is FUT8 deficient or thathas a mutated version of FUT8. Low fucose or defucosylated antibodiesand functional fragment have increased ADCC activity. In addition toalterations in fucose, the disclosed antibodies and functional fragmentsmay comprise other functional modifications to their glycosylationpatterns. For instance, modifications at position 297 (e.g. N297A andN297Q) can prevent glycosylation of the Fc region altogether, thuseliminating Fc function, ADCC, and CDC.

In some embodiments, the anti-PD-L1 antibody is CTI-07, CTI-09, CTI-48,CTI-49, CTI-50, CTI-76, CTI-77, CTI-78, CTI-57, or CTI-58 or afunctional fragment thereof. Tables 1 and 2 provides exemplary CDRsequences of the disclosed anti-PD-L1 antibodies and functionalfragments thereof.

TABLE 1 Anitbody HCDR1 HCDR2 HCDR3 CTI-48 GTFSRSAIS VIIPAFGEANYAQKFQGARGRQMFGAGIDF (SEQ ID NO: 2) (SEQ ID NO: 9) (SEQ ID NO: 15) CTI-49GTFSGYAIS VIIPAFGTANYAQKFQG ARGRQMFGAGIDF (SEQ ID NO: 3) (SEQ ID NO: 10)(SEQ ID NO: 15) CTI-76 GTFWRYAIS VIIPIWGKANYAQKFQG ARGRQMFGAGIDF(SEQ ID NO: 4) (SEQ ID NO: 11) (SEQ ID NO: 15) CTI-77 GTGGSYAISGIYPAFGTANYAQKFQG ARGRQMFGAGIDF (SEQ ID NO: 5) (SEQ ID NO: 12)(SEQ ID NO: 15) CTI-78 GTFGTYAIS GIYPRFGTANYAQKFQG ARGRQMFGAGIDF(SEQ ID NO: 6) (SEQ ID NO: 13) (SEQ ID NO: 15) CTI-50 GTFSPKAISVIIPIFGPANYAKFQG ARGRQMFGAGIDF (SEQ ID NO: 7) (SEQ ID NO: 14)(SEQ ID NO: 15) CTI-57 YTLSSHGIT WISAHSGHASNAQKVED ARVWRALYHGMDV(SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO: 18) CTI-58 YTLSSHGITWISAHSGHASNAQKVED ARVHAALYHGMDV (SEQ ID NO: 16) (SEQ ID NO: 17)(SEQ ID NO: 19) CTI-09 GTFSSYAIS GIIPIFGTANYAQKFQG ARGRQMFGAGIDF(SEQ ID NO: 1) (SEQ ID NO: 8) (SEQ ID NO: 15) CTI-07 YTLSSHGITWISAHSGHASNAQKVED ARVHAALYYGMDV (SEQ ID NO: 16) (SEQ ID NO: 17)(SEQ ID NO: 57) CTI-97 GTFSRSAIS VIIPAFGEANYAQKFQG ARGRQMFGAGIDF(SEQ ID NO: 2) (SEQ ID NO: 9) (SEQ ID NO: 15) CTI-98 GTFSRSAISVIIPAFGEANYAQKFQG ARGRQMFGAGIDF (SEQ ID NO: 2) (SEQ ID NO: 9)(SEQ ID NO: 15) CTI-92 GTFSRSAIS VIIPAFGEANYAQKFQG ARGRQMFGAGIDF(SEQ ID NO: 2) (SEQ ID NO: 9) (SEQ ID NO: 15) CTI-95 GTFSRSAISVIIPAFGEANYAQKFQG ARGRQMFGAGIDF (SEQ ID NO: 2) (SEQ ID NO: 9)(SEQ ID NO: 15) CTI-93 GTFSRSAIS VIIPAFGEANYAQKFQG ARGRQMFGAGIDF(SEQ ID NO: 2) (SEQ ID NO: 9) (SEQ ID NO: 15) CTI-94 GTFSRSAISVIIPAFGEANYAQKFQG ARGRQMFGAGIDF (SEQ ID NO: 2) (SEQ ID NO: 9)(SEQ ID NO: 15) CTI-96 GTFSRSAIS VIIPAFGEANYAQKFQG ARGRQMFGAGIDF(SEQ ID NO: 2) (SEQ ID NO: 9) (SEQ ID NO: 15)

TABLE 2 Anitbody LCDR1 LCDR2 LCDR3 CTI-48 TRSSGSIDSNYVQ EDNQRPSQSYDSNNRHVI (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) CTI-49TRSSGSIDSNYVQ EDNQRPS QSYDSNNRHVI (SEQ ID NO: 20) (SEQ ID NO: 21)(SEQ ID NO: 22) CTI-76 TRSSGSIDSNYVQ EDNQRPS QSYDSNNRHVI (SEQ ID NO: 20)(SEQ ID NO: 21) (SEQ ID NO: 22) CTI-77 TRSSGSIDSNYVQ EDNQRPS QSYDSNNRHVI(SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22) CTI-78 TRSSGSIDSNYVQEDNQRPS QSYDSNNRHVI (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22)CTI-50 TRSSGSIDSNYVQ EDNQRPS QSYDSNNRHVI (SEQ ID NO: 20) (SEQ ID NO: 21)(SEQ ID NO: 22) CTI-57 GGNNIGSKGVH DDSDRPS QVWDSSSDHWV (SEQ ID NO: 23)(SEQ ID NO: 24) (SEQ ID NO: 25) CTI-58 GGNNIGSKGVH DDSDRPS QVWDSSSDHWV(SEQ ID NO: 23) (SEQ ID NO: 24) (SEQ ID NO: 25) CTI-09 TRSSGSIDSNYVQEDNQRPS QSYDSNNRHVI (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22)CTI-07 GGNNIGSKGVH DDSDRPS QVWDSSSDHWV (SEQ ID NO: 23) (SEQ ID NO: 24)(SEQ ID NO: 25) CTI-97 TRSSGSIDSNYVQ EDNQRPS QSYDSNLRHVI (SEQ ID NO: 20)(SEQ ID NO: 21) (SEQ ID NO: 75) CTI-98 TRSSGSIDSNYVQ EDNQRPS QSYDSNLRHVI(SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 75) CTI-92 TRSSGSIDSNYVQEDNQRPS QSYDSNNRHVI (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 22)CTI-95 TRSSGSIDSNYVQ EDNQRPS QSYDSNNRHVI (SEQ ID NO: 20) (SEQ ID NO: 21)(SEQ ID NO: 22) CTI-93 TRSSGSIDSNYVQ EDNQRPS QSYDSNIRHVI (SEQ ID NO: 20)(SEQ ID NO: 21) (SEQ ID NO: 76) CTI-94 TRSSGSIDSNYVQ EDNQRPS QSYDSNLRHVI(SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 75) CTI-96 TRSSGSIDSNYVQEDNQRPS QSYDSNIRHVI (SEQ ID NO: 20) (SEQ ID NO: 21) (SEQ ID NO: 76)

Additionally, the disclosed antibodies and functional fragments may alsocomprise various framework regions. For instance, in some embodimentsthe disclosed antibodies and functional fragments comprise SEQ ID NOs26-34 and/or 43-47. In some embodiments, certain alterations to theframework regions may be particularly advantageous. For instance,substituting glutamic acid (E) for glutamine (Q) in the first positionof framework region one of the heavy chain of an antibody can increasemanufacturing product stability efficiency. Accordingly, someembodiments of the disclosed antibodies and fragments will incorporatethis modification. Thus, in some embodiments, the heavy chain of thedisclosed antibodies or functional fragments will comprise SEQ ID NOs:36-41, while in other embodiments, the heavy chain of the disclosedantibodies or functional fragments will comprise SEQ ID NOs: 48-49 or51-56. Furthermore, in some embodiments, the heavy chain of thedisclosed antibodies or functional fragments will comprise SEQ ID NOs:71-72, or polypeptide encoded by a nucleic acid sequence comprising SEQID NOs: 59-68.

In some embodiments, the light chain of the disclosed antibodies orfunctional fragments will comprise SEQ ID NOs: 42 or 50. Furthermore, insome embodiments, the light chain of the disclosed antibodies orfunctional fragments will comprise a polypeptide encoded by a nucleicacid sequence comprising SEQ ID NOs: 69-70.

One of ordinary skill in the art will understand that certain changescan be made to the disclosed sequences without compromising the bindingaffinity or function of the disclosed anti-PD-L1 antibodies andfunctional fragments. According, in some embodiments, the anti-PD-L1antibodies or functional fragments will share about 80%, about 81%,about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%,about 95%, about 96%, about 97%, about 98%, or about 99% sequenceidentity with the disclosed sequences.

In some embodiments, the disclosure provides for isolated nucleic acidsequences encoding an anti-PD-L1 antibody or functional fragmentthereof, for examples, SEQ ID NOs: 59-70.

The disclosed antibodies and functional fragments thereof may be definedby sequence, or by functional characteristics. For instance, thedisclosed antibodies and functional fragments thereof, can have a K_(D)of at least 3.0×10⁻⁸, at least 2.5×10⁻⁸, at least 2.0×10⁻⁸, at least1.5×10⁻⁸, at least 1.0×10⁻⁸, at least 0.5×10⁻⁸, at least 9.95×10⁻⁹, atleast 9.90×10⁻⁹, at least 9.85×10⁻⁹, at least 9.80×10⁻⁹, at least9.75×10⁻⁹, at least 9.70×10⁻⁹, at least 9.65×10⁻⁹, at least 9.60×10⁻⁹,at least 9.55×10⁻⁹, at least 9.5×10⁻⁹, at least 9.45×10⁻⁹, at least9.40×10⁻⁹, at least 9.35×10⁻⁹, at least 9.30×10⁻⁹, at least 9.25×10⁻⁹,at least 9.20×10⁻⁹, at least 9.15×10⁻⁹, at least 9.10×10⁻⁹, at least9.05×10⁻⁹, at least 9.0×10⁻⁹, at least 8.95×10⁻⁹, at least 8.90×10⁻⁹, atleast 8.85×10⁻⁹, at least 8.80×10⁻⁹, at least 8.75×10⁻⁹, at least8.70×10⁻⁹, at least 8.65×10⁻⁹, at least 8.60×10⁻⁹, at least 8.55×10⁻⁹,at least 8.5×10⁻⁹, at least 8.45×10⁻⁹, at least 8.40×10⁻⁹, at least8.35×10⁻⁹, at least 8.30×10⁻⁹, at least 8.25×10⁻⁹, at least 8.20×10⁻⁹,at least 8.15×10⁻⁹, at least 8.10×10⁻⁹, at least 8.05×10⁻⁹, at least8.0×10⁻⁹, at least 7.95×10⁻⁹, at least 7.90×10⁻⁹, at least 7.85×10⁻⁹, atleast 7.80×10⁻⁹, at least 7.75×10⁻⁹, at least 7.70×10⁻⁹, at least7.65×10⁻⁹, at least 7.60×10⁻⁹, at least 7.55×10⁻⁹, at least 7.5×10⁻⁹, atleast 7.45×10⁻⁹, at least 7.40×10⁻⁹, at least 7.35×10⁻⁹, at least7.30×10⁻⁹, at least 7.25×10⁻⁹, at least 7.20×10⁻⁹, at least 7.15×10⁻⁹,at least 7.10×10⁻⁹, at least 7.05×10⁻⁹, at least 7.0×10⁻⁹, at least6.95×10⁻⁹, at least 6.90×10⁻⁹, at least 6.85×10⁻⁹, at least 6.80×10⁻⁹,at least 6.75×10⁻⁹, at least 6.70×10⁻⁹, at least 6.65×10⁻⁹, at least6.60×10⁻⁹, at least 6.55×10⁻⁹, at least 6.5×10⁻⁹, at least 6.45×10⁻⁹, atleast 6.40×10⁻⁹, at least 6.35×10⁻⁹, at least 6.30×10⁻⁹, at least6.25×10⁻⁹, at least 6.20×10⁻⁹, at least 6.15×10⁻⁹, at least 6.10×10⁻⁹,at least 6.05×10⁻⁹, at least 6.0×10⁻⁹, at least 5.95×10⁻⁹, at least5.90×10⁻⁹, at least 5.85×10⁻⁹, at least 5.80×10⁻⁹, at least 5.75×10⁻⁹,at least 5.70×10⁻⁹, at least 5.65×10⁻⁹, at least 5.60×10⁻⁹, at least5.55×10⁻⁹, at least 5.5×10⁻⁹, at least 5.45×10⁻⁹, at least 5.40×10⁻⁹, atleast 5.35×10⁻⁹, at least 5.30×10⁻⁹, at least 5.25×10⁻⁹, at least5.20×10⁻⁹, at least 5.15×10⁻⁹, at least 5.10×10⁻⁹, at least 5.05×10⁻⁹,at least 5.0×10⁻⁹, at least 4.95×10⁻⁹, at least 4.90×10⁻⁹, at least4.85×10⁻⁹, at least 4.80×10⁻⁹, at least 4.75×10⁻⁹, at least 4.70×10⁻⁹,at least 4.65×10⁻⁹, at least 4.60×10⁻⁹, at least 4.55×10⁻⁹, at least4.5×10⁻⁹, at least 4.45×10⁻⁹, at least 4.40×10⁻⁹, at least 4.35×10⁻⁹, atleast 4.30×10⁻⁹, at least 4.25×10⁻⁹, at least 4.20×10⁻⁹, at least4.15×10⁻⁹, at least 4.10×10⁻⁹, at least 4.05×10⁻⁹, at least 4.0×10⁻⁹, atleast 3.95×10⁻⁹, at least 3.90×10⁻⁹, at least 3.85×10⁻⁹, at least3.80×10⁻⁹, at least 3.75×10⁻⁹, at least 3.70×10⁻⁹, at least 3.65×10⁻⁹,at least 3.60×10⁻⁹, at least 3.55×10⁻⁹, at least 3.5×10⁻⁹, at least3.45×10⁻⁹, at least 3.40×10⁻⁹, at least 3.35×10⁻⁹, at least 3.30×10⁻⁹,at least 3.25×10⁻⁹, at least 3.20×10⁻⁹, at least 3.15×10⁻⁹, at least3.10×10⁻⁹, at least 3.05×10⁻⁹, at least 3.0×10⁻⁹, at least 2.95×10⁻⁹, atleast 2.90×10⁻⁹, at least 2.85×10⁻⁹, at least 2.80×10⁻⁹, at least2.75×10⁻⁹, at least 2.70×10⁻⁹, at least 2.65×10⁻⁹, at least 2.60×10⁻⁹,at least 2.55×10⁻⁹, at least 2.5×10⁻⁹, at least 2.45×10⁻⁹, at least2.40×10⁻⁹, at least 2.35×10⁻⁹, at least 2.30×10⁻⁹, at least 2.25×10⁻⁹,at least 2.20×10⁻⁹, at least 2.15×10⁻⁹, at least 2.10×10⁻⁹, at least2.05×10⁻⁹, at least 2.0×10⁻⁹, at least 1.95×10⁻⁹, at least 1.90×10⁻⁹, atleast 1.85×10⁻⁹, at least 1.80×10⁻⁹, at least 1.75×10⁻⁹, at least1.70×10⁻⁹, at least 1.65×10⁻⁹, at least 1.60×10⁻⁹, at least 1.55×10⁻⁹,at least 1.5×10⁻⁹, at least 1.45×10⁻⁹, at least 1.40×10⁻⁹, at least1.35×10⁻⁹, at least 1.30×10⁻⁹, at least 1.25×10⁻⁹, at least 1.20×10⁻⁹,at least 1.15×10⁻⁹, at least 1.10×10⁻⁹, at least 1.05×10⁻⁹, at least1.0×10⁻⁹, at least 0.95×10⁻⁹, at least 0.90×10⁻⁹, at least 0.85×10⁻⁹, atleast 0.80×10⁻⁹, at least 0.75×10⁻⁹, at least 0.70×10⁻⁹, at least0.65×10⁻⁹, at least 0.60×10⁻⁹, at least 0.55×10⁻⁹, at least 0.5×10⁻⁹, atleast 0.45×10⁻⁹, at least 0.40×10⁻⁹, at least 0.35×10⁻⁹, at least0.30×10⁻⁹, at least 0.25×10⁻⁹, at least 0.20×10⁻⁹, at least 0.15×10⁻⁹,at least 0.10×10⁻⁹, at least 0.05×10⁻⁹, at least 9.5×10⁻¹⁰, at least9.0×10⁻¹⁰, at least 8.5×10⁻¹⁰, at least 8.0×10⁻¹⁰, or any value inbetween. For example, the disclosed antibodies and functional fragmentsthereof can have K_(D) values of 8.2×10⁻¹⁰, 2.31×10⁻⁰⁹, 8.24×10⁻⁰⁹,3.25×10⁻⁰⁹, 3.46×10⁻⁰⁹, 1.91×10⁻⁰⁹, 7.97×10⁻⁰⁸, 2.41×10⁻⁰⁸, 9.5×10⁻¹⁰,or 8.6×10⁻¹⁰.

Likewise, the disclosed antibodies and functional fragments thereof canhave IC₅₀ values between 4.0×10⁻⁵ μg/ml and 9.5×10⁻⁷ μg/ml or any valuein between. For example, the disclosed antibodies and functionalfragments thereof can have IC₅₀ values of 9.19×10⁻⁷, 4.156×10⁻⁵,9.985×10⁻⁷, 1.037×10⁻⁶ or 3.463×10⁻⁶.

The anti-PD-L1 antibody or a PD-L1-binding functional fragment thereofcan be formulated in a pharmaceutical composition suitable foradministration to the target subject by the intended route ofadministration, as discussed in more detail below.

III. Pharmaceutical Compositions and Formulations

Pharmaceutical compositions suitable for use in the methods describedherein can include the therapeutically active agent (e.g., anti-PD-L1antibodies or functional fragments thereof) and a pharmaceuticallyacceptable carrier or diluent.

The composition may be formulated for intravenous, subcutaneous,intraperitoneal, intramuscular, oral, nasal, pulmonary, ocular, vaginal,or rectal administration. In some embodiments, anti-PD-L1 antibodies orfunctional fragments thereof are formulated for intravenous,subcutaneous, intraperitoneal, or intramuscular administration, such asin a solution, suspension, emulsion, liposome formulation, etc. Thepharmaceutical composition can be formulated to be an immediate-releasecomposition, sustained-release composition, delayed-release composition,etc., using techniques known in the art.

Pharmacologically acceptable carriers for various dosage forms are knownin the art. For example, excipients, lubricants, binders, anddisintegrants for solid preparations are known; solvents, solubilizingagents, suspending agents, isotonicity agents, buffers, and soothingagents for liquid preparations are known. In some embodiments, thepharmaceutical compositions include one or more additional components,such as one or more preservatives, antioxidants, stabilizing agents andthe like.

Additionally, the disclosed pharmaceutical compositions can beformulated as a solution, microemulsion, liposome, or other orderedstructure suitable to high drug concentration. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. In some embodiment, it willbe preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent that delaysabsorption, for example, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Pharmaceutical compositions of the disclosure can be administered incombination with other therapeutics. For example, the combinationtherapy can include a pharmaceutical composition comprising at least oneof the disclosed anti-PD-L1 antibodies or functional fragments thereofwith at least one or more additional therapeutic agents, including butnot limited to, CAR T cells (e.g., modified T cells that express ananti-CD19, anti-Her2, anti-BCMA, anti-CS-1, anti-PSCA, anti-CAIX,anti-IL13R, or anti-PD-L1 CAR), other tumor-targeting antibodies (e.g.,an anti-CAIX antibody), immune response potentiating modalities (e.g.,an anti-GITR antibody, an anti-OX40 antibody, an anti-CD137 antibody, ora TLR agonist), and small molecule drugs (e.g., a BTK inhibitor, an EGFRinhibitor, a BET inhibitor, a PI3Kdelta inhibitor, a BRAF inhibitor, ora PARP inhibitor). The pharmaceutical compositions of the disclosure canalso be administered in conjunction with radiation therapy.

IV. Methods of Treating Cancer

Provided herein are methods of treating cancer, malignant disease, orcancer cell proliferation with the disclosed anti-PD-L1 antibodies. Morespecifically, the disclosure provides for methods of enhancing T-cellfunction and anti-tumor immunity comprising administering atherapeutically effective amount of any of the above describedanti-PD-L1 antibodies or compositions.

Enhancing T-cell function and anti-tumor immunity provides a broadspectrum approach to treating cancer, malignant disease, or cancer cellproliferation. Accordingly, numerous types of cancer can be treated byadministering the disclosed anti-PD-L1 antibodies and functionalfragments thereof. For example, in some embodiments, the cancer is ahematological cancer (e.g., lymphoma, Non-Hodgkin's lymphoma, chroniclymphocytic leukemia, or multiple myeloma), a neurological cancer,breast cancer, a gastrointestinal cancer (e.g., colon cancer), renalcell carcinoma (e.g., clear cell renal cell carcinoma), or agenitourinary cancer (e.g., ovarian cancer). In some embodiments, thecancer is melanoma, lung cancer (e.g., non-small cell lung cancer), headand neck cancer, liver cancer, pancreatic cancer, bone cancer, prostatecancer, bladder cancer, or a vascular cancer.

In some embodiments, the cancer being treated according to the disclosedmethods is a highly immunogenic carcinoma. And in some embodiments, thecancer being treated according to the disclosed methods is a cancer thatexpresses PD-L1.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response like tumor regression or remission). Forexample, in some embodiments, a single bolus may be administered, whilein some embodiments, several divided doses may be administered over timeor the dose may be proportionally reduced or increased as indicated bythe situation. For example, in some embodiments the disclosed antibodiesor functional fragments may be administered once or twice weekly bysubcutaneous or intravenous injection. In some embodiments, thedisclosed antibodies or functional fragments may be administered once ortwice monthly by subcutaneous injection. In some embodiments, thedisclosed antibodies or functional fragments may be administered onceevery week, once every other week, once every three weeks, once everyfour weeks, once every other month, once every three months, once everyfour months, once every five months, or once every six months.

Exemplary doses can vary according to the size and health of theindividual being treated, as well as the condition being treated. Forexample, in some embodiments, the disclosed antibodies or functionalfragments may be administered in a dose of 1-100 mg/kg. In someembodiments, the disclosed antibodies and functional fragments may beadministered in a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, or 100 mg/kg.

Furthermore, the disclosed methods of treatment can additionallycomprise the administration of a second therapeutic compound in additionto the anti-PD-L1 antibody or functional fragment thereof. For example,in some embodiments, the additional therapeutic compound is a CAR-Tcell, a tumor-targeting antibody, an immune response potentiatingmodality, or a small molecule drug.

In some embodiments, the disclosed methods may utilize antibodies andfunctional fragments thereof with various functional characteristics.For instance, the disclosed methods can comprise anti-PD-L1 antibodiesand functional fragments thereof, having a K_(D) of at least 3.0×10⁻⁸,at least 2.5×10⁻⁸, at least 2.0×10⁻⁸, at least 1.5×10⁻⁸, at least1.0×10⁻⁸, at least 0.5×10⁻⁸, at least 9.95×10⁻⁹, at least 9.90×10⁻⁹, atleast 9.85×10⁻⁹, at least 9.80×10⁻⁹, at least 9.75×10⁻⁹, at least9.70×10⁻⁹, at least 9.65×10⁻⁹, at least 9.60×10⁻⁹, at least 9.55×10⁻⁹,at least 9.5×10⁻⁹, at least 9.45×10⁻⁹, at least 9.40×10⁻⁹, at least9.35×10⁻⁹, at least 9.30×10⁻⁹, at least 9.25×10⁻⁹, at least 9.20×10⁻⁹,at least 9.15×10⁻⁹, at least 9.10×10⁻⁹, at least 9.05×10⁻⁹, at least9.0×10⁻⁹, at least 8.95×10⁻⁹, at least 8.90×10⁻⁹, at least 8.85×10⁻⁹, atleast 8.80×10⁻⁹, at least 8.75×10⁻⁹, at least 8.70×10⁻⁹, at least8.65×10⁻⁹, at least 8.60×10⁻⁹, at least 8.55×10⁻⁹, at least 8.5×10⁻⁹, atleast 8.45×10⁻⁹, at least 8.40×10⁻⁹, at least 8.35×10⁻⁹, at least8.30×10⁻⁹, at least 8.25×10⁻⁹, at least 8.20×10⁻⁹, at least 8.15×10⁻⁹,at least 8.10×10⁻⁹, at least 8.05×10⁻⁹, at least 8.0×10⁻⁹, at least7.95×10⁻⁹, at least 7.90×10⁻⁹, at least 7.85×10⁻⁹, at least 7.80×10⁻⁹,at least 7.75×10⁻⁹, at least 7.70×10⁻⁹, at least 7.65×10⁻⁹, at least7.60×10⁻⁹, at least 7.55×10⁻⁹, at least 7.5×10⁻⁹, at least 7.45×10⁻⁹, atleast 7.40×10⁻⁹, at least 7.35×10⁻⁹, at least 7.30×10⁻⁹, at least7.25×10⁻⁹, at least 7.20×10⁻⁹, at least 7.15×10⁻⁹, at least 7.10×10⁻⁹,at least 7.05×10⁻⁹, at least 7.0×10⁻⁹, at least 6.95×10⁻⁹, at least6.90×10⁻⁹, at least 6.85×10⁻⁹, at least 6.80×10⁻⁹, at least 6.75×10⁻⁹,at least 6.70×10⁻⁹, at least 6.65×10⁻⁹, at least 6.60×10⁻⁹, at least6.55×10⁻⁹, at least 6.5×10⁻⁹, at least 6.45×10⁻⁹, at least 6.40×10⁻⁹, atleast 6.35×10⁻⁹, at least 6.30×10⁻⁹, at least 6.25×10⁻⁹, at least6.20×10⁻⁹, at least 6.15×10⁻⁹, at least 6.10×10⁻⁹, at least 6.05×10⁻⁹,at least 6.0×10⁻⁹, at least 5.95×10⁻⁹, at least 5.90×10⁻⁹, at least5.85×10⁻⁹, at least 5.80×10⁻⁹, at least 5.75×10⁻⁹, at least 5.70×10⁻⁹,at least 5.65×10⁻⁹, at least 5.60×10⁻⁹, at least 5.55×10⁻⁹, at least5.5×10⁻⁹, at least 5.45×10⁻⁹, at least 5.40×10⁻⁹, at least 5.35×10⁻⁹, atleast 5.30×10⁻⁹, at least 5.25×10⁻⁹, at least 5.20×10⁻⁹, at least5.15×10⁻⁹, at least 5.10×10⁻⁹, at least 5.05×10⁻⁹, at least 5.0×10⁻⁹, atleast 4.95×10⁻⁹, at least 4.90×10⁻⁹, at least 4.85×10⁻⁹, at least4.80×10⁻⁹, at least 4.75×10⁻⁹, at least 4.70×10⁻⁹, at least 4.65×10⁻⁹,at least 4.60×10⁻⁹, at least 4.55×10⁻⁹, at least 4.5×10⁻⁹, at least4.45×10⁻⁹, at least 4.40×10⁻⁹, at least 4.35×10⁻⁹, at least 4.30×10⁻⁹,at least 4.25×10⁻⁹, at least 4.20×10⁻⁹, at least 4.15×10⁻⁹, at least4.10×10⁻⁹, at least 4.05×10⁻⁹, at least 4.0×10⁻⁹, at least 3.95×10⁻⁹, atleast 3.90×10⁻⁹, at least 3.85×10⁻⁹, at least 3.80×10⁻⁹, at least3.75×10⁻⁹, at least 3.70×10⁻⁹, at least 3.65×10⁻⁹, at least 3.60×10⁻⁹9,at least 3.55×10⁻⁹, at least 3.5×10⁻⁹, at least 3.45×10⁻⁹, at least3.40×10⁻⁹, at least 3.35×10⁻⁹, at least 3.30×10⁻⁹, at least 3.25×10⁻⁹,at least 3.20×10⁻⁹, at least 3.15×10⁻⁹, at least 3.10×10⁻⁹, at least3.05×10⁻⁹, at least 3.0×10⁻⁹, at least 2.95×10⁻⁹, at least 2.90×10⁻⁹, atleast 2.85×10⁻⁹, at least 2.80×10⁻⁹, at least 2.75×10⁻⁹, at least2.70×10⁻⁹, at least 2.65×10⁻⁹, at least 2.60×10⁻⁹, at least 2.55×10⁻⁹,at least 2.5×10⁻⁹, at least 2.45×10⁻⁹, at least 2.40×10⁻⁹, at least2.35×10⁻⁹, at least 2.30×10⁻⁹, at least 2.25×10⁻⁹, at least 2.20×10⁻⁹,at least 2.15×10⁻⁹, at least 2.10×10⁻⁹, at least 2.05×10⁻⁹, at least2.0×10⁻⁹9, at least 1.95×10⁻⁹, at least 1.90×10⁻⁹, at least 1.85×10⁻⁹,at least 1.80×10⁻⁹, at least 1.75×10⁻⁹, at least 1.70×10⁻⁹, at least1.65×10⁻⁹, at least 1.60×10⁻⁹, at least 1.55×10⁻⁹, at least 1.5×10⁻⁹, atleast 1.45×10⁻⁹, at least 1.40×10⁻⁹, at least 1.35×10⁻⁹, at least1.30×10⁻⁹, at least 1.25×10⁻⁹, at least 1.20×10⁻⁹, at least 1.15×10⁻⁹,at least 1.10×10⁻⁹, at least 1.05×10⁻⁹, at least 1.0×10⁻⁹, at least0.95×10⁻⁹, at least 0.90×10⁻⁹, at least 0.85×10⁻⁹, at least 0.80×10⁻⁹,at least 0.75×10⁻⁹, at least 0.70×10⁻⁹, at least 0.65×10⁻⁹, at least0.60×10⁻⁹, at least 0.55×10⁻⁹, at least 0.5×10⁻⁹, at least 0.45×10⁻⁹, atleast 0.40×10⁻⁹, at least 0.35×10⁻⁹, at least 0.30×10⁻⁹, at least0.25×10⁻⁹, at least 0.20×10⁻⁹, at least 0.15×10⁻⁹, at least 0.10×10⁻⁹,at least 0.05×10⁻⁹, at least 9.5×10⁻¹⁰, at least 9.0×10⁻¹⁰, at least8.5×10⁻¹⁰, at least 8.0×10⁻¹⁰, or any value in between. For example, thedisclosed methods can comprise anti-PD-L1 antibodies and functionalfragments thereof having K_(D) values of 8.2×10⁻¹⁰, 2.31×10⁻⁰⁹,8.24×10⁻⁰⁹, 3.25×10⁻⁰⁹, 3.46×10⁻⁰⁹, 1.91×10⁻⁰⁹, 7.97×10⁻⁰⁸, 2.41×10⁻⁰⁸,9.5×10⁻¹⁰, or 8.6×10⁻¹⁰.

Likewise, the disclosed methods can comprise anti-PD-L1 antibodies andfunctional fragments thereof having IC₅₀ values between 4.0×10⁻⁵ μg/mland 9.5×10⁻⁷ μg/ml or any value in between. For example, the disclosedmethods can comprise anti-PD-L1 antibodies and functional fragmentsthereof having IC₅₀ values of 9.19×10⁻⁷, 4.156×10⁻⁵, 9.985×10⁻⁷,1.037×10⁻⁶, or 3.463×10⁻⁶.

In some embodiments, an immune response potentiating modality cancomprise an anti-GITR antibody, an anti-OX40 antibody, an anti-CD137antibody, a TLR agonist, or anti-CD40 antibody.

In some embodiments, a tumor-targeting antibody can comprise ananti-CAIX antibody, an anti-BCMA, an anti-CS-1, an anti-CD20 (e.g.Ublituximab), an anti-Her2, an anti-PCSA, or an anti-FcRL5.

In some embodiments, a small molecule drug can comprise atumor-targeting small molecule drug, or, for instance, a BTK inhibitor(e.g. ibrutinib), an EGFR inhibitor (e.g. CK-101), a BET inhibitor (e.g.CK-103), a PARP inhibitor (e.g. olaparib or CK-102), a PI3Kdeltainhibitor (e.g. TGR-1202), or a BRAF inhibitor (e.g. Vemurafenib).

Particular treatment regimens may be evaluated according to whether itwill improve a given patient's outcome, meaning it will reduce the riskof recurrence or increase the likelihood of progression-free survival ofthe given cancer.

Thus, for the purposes of this disclosure, a subject is treated if oneor more beneficial or desired results, including desirable clinicalresults, are obtained. For example, beneficial or desired clinicalresults include, but are not limited to, one or more of the following:decreasing one or more symptoms resulting from the disease, increasingthe quality of life of those suffering from the disease, decreasing thedose of other medications required to treat the disease, delaying theprogression of the disease, and/or prolonging survival of individuals.

Furthermore, while the subject of the methods is generally a cancerpatient, the age of the patient is not limited. The disclosed methodsare useful for treating cancer, malignant disease, or cancer cellproliferation with various recurrence and prognostic outcomes across allage groups and cohorts. Thus, in some embodiments, the subject may be apediatric subject, while in other embodiments, the subject may be anadult subject.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.

EXAMPLES Example 1—Treatment of a Cancer Patient with the DisclosedAnti-PD-L1 Antibodies

This example illustrates methods using anti-PD-L1 antibodies in thetreatment of cancer.

A patient known to have or suspected of having cancer is administered atherapeutically effective amount of a pharmaceutical compositioncomprising an anti-PD-L1 antibody, by intravenous or subcutaneousinjection. The patient is evaluated for the presence and/or severity ofsigns and symptoms associated with cancer, including, but not limitedto, pain, weakness, etc., and the patient is treated until one or moresigns/symptoms is reduced, ameliorated, or eliminated. Optionally,samples may be taken from the patient to monitor cancer progressionfollowing treatment. Optionally, another dose of the pharmaceuticalcomposition is administered if signs/symptoms persist and/or if thecancer progresses or recurs.

Example 2—Production of Optimized Antibodies

Antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotinylation Kitfrom Pierce. Goat anti-human F(ab′)2 kappa-FITC (LC-FITC), Extravidin-PE(EA-PE) and streptavidin-633 (SA-633) were obtained from SouthernBiotech, Sigma and Molecular Probes, respectively. StreptavidinMicroBeads and MACS LC separation columns were purchased from MiltenyiBiotec.

Affinity Maturation

Binding optimization of naïve clones was carried out utilizing threematuration strategies: diversification of VH CDRH1/CDRH2, PCRmutagenesis of the VH gene and VH mutagenesis with a focus on CDRH3.

CTI-07 Lineage

The first cycle of optimization focused on selection of improved bindersfrom a library in which the CTI-07 VH gene was diversified by mutagenicPCR using techniques known in the art. Round 1: Selections wereperformed by presenting VH mutated forms of the full-length CTI-07 IgGon the surface of yeast. These libraries were incubated with 100 nMbiotinylated PD-L1, then detecting IgG expression by an anti-LC FITCreagent (IgG expression) and SA-633 (detection of antigen binding) andviable cells by propidium iodine staining. The top antigen binding/IgGexpressing cells were selected by FACS. Round 2: Selections wereperformed as per Round 1, but using 10 nM biotinylated PD-L1 fordiscrimination of antigen binding. Round 3: Library expression wascarried out as per Rounds 1 &2. Round 3 employed the use of apoly-specificity reagent (PSR) to remove non-specific antibodies fromthe selection output (Y. Xu et. al., “Addressing Polyspecificity ofAntibodies Selected from an in vitro Yeast Presentation System: aFACS-based, High-Throughput Selection and Analytical Tool.” PEDS 26.10(2013): 663-70.) These libraries were incubated with 1:10 dilution ofbiotinylated PSR reagent, IgG expression was detected by an anti-LC FITCreagent (IgG expression) and PSR binding was detected by EA-PE(detection of antigen binding) and viable cells by propidium iodinestaining. The top 1-2% of IgG positive, PSR negative, PI negative cellswere sorted and carried to Round 4. Round 4: Selections were performedas per Round 2, but using 1 nM biotinylated PD-L1 for discrimination ofantigen binding. Top clones were plated, and sequenced to determineunique IgG sequences. Unique IgG sequences were submitted for antibodyproduction, purification and characterization.

The second cycle of optimization focused on the selection of improvedbinders from a library in which the VH gene was diversified by mutagenicPCR while also utilizing degenerate CDRH3 oligos to increase themutagenic rate within CDRH3. This amplification technique was performedusing techniques well known in the art. Round 1: Selections wereperformed by presenting VH mutated forms of the full-length parent IgGon the surface of yeast. These libraries were incubated with 10 nMbiotinylated PD-L1, then detecting IgG expression by an anti-LC FITCreagent (IgG expression) and SA-633 (detection of antigen binding) andviable cells by propidium iodine staining. The top antigen binding/IgGexpressing cells were selected by FACS. Round 2: Selections wereperformed as per Round 1, but using 2 nM biotinylated PD-L1 fordiscrimination of antigen binding. Top clones were plated, and sequencedto determine unique IgG sequences. Unique IgG sequences were submittedfor antibody production, purification and characterization.

CTI-09 Lineage

CTI-09 optimization employed the use of CDRH1 and CDRH2 variegation: TheCDRH3 of CTI-09 was amplified by PCR and then recombined into a premadevector library with CDRH1 and CDRH2 variants of a diversity of 1×108.Round 1: Selections were performed by presenting VH mutated forms of thefull-length CTI-09 IgG on the surface of yeast. These libraries wereincubated with 100 nM biotinylated PD-L1. Antigen positive cells wereselected by magnetic separation via the Miltenyi MACS system. In shortlibraries incubated with b-PD-L1 were incubated with streptavidinmagnetic beads. Yeast/bead complexes were captured on a MACS LS column,with unlabeled cells passing into the waste. b-PD-L1 binding cells werethen eluted into media for propagation for Round 2 of the selectionprocess. Round 2: Selections were performed by presenting VH mutatedforms of the full-length CTI-07 IgG on the surface of yeast. Theselibraries were incubated with 20 nM biotinylated PD-L1, then detectingIgG expression by an anti-LC FITC reagent (IgG expression) and SA-633(detection of antigen binding) and viable cells by propidium iodinestaining. The top antigen binding/IgG expressing cells were selected byFACS. Round 3: Library expression was carried out as per Rounds 1&2.Round 3 employed the use of a poly-specificity reagent (PSR) to removenon-specific antibodies from the selection output (Y. Xu et. al.,“Addressing Polyspecificity of Antibodies Selected from an in vitroYeast Presentation System: a FACS-based. High-Throughput Selection andAnalytical Tool.” PEDS 26.10 (2013): 663-70.) These libraries wereincubated with 1:10 dilution of biotinylated PSR reagent, IgG expressionwas detected by an anti-LC FITC reagent (IgG expression) and PSR bindingwas detected by EA-PE (detection of antigen binding) and viable cells bypropidium iodine staining. The top 1-2% of IgG positive, PSR negative,PI negative cells were sorted and carried to Round 4. Round 4: Theinduced Round 3 output was incubated with 20 nM b-PD-L1. Cells werepelleted and washed to remove any remaining b-PD-L1. This cell pelletwas resuspended in 1 uM unlabeled PD-L1. Top antigen binders werediscriminated by their ability to retain b-PD-L1 antigen over time. Topclones were plated, and sequenced to determine unique IgG sequences.Unique IgG sequences were submitted for antibody production,purification and characterization.

Antibody Production and Purification

Yeast clones were grown to saturation and then induced for 48 h at 30°C. with shaking. After induction, yeast cells were pelleted and thesupernatants were harvested for purification. IgGs were purified using aProtein A column and eluted with acetic acid, pH 2.0. Fab fragments weregenerated by papain digestion and purified over KappaSelect (GEHealthcare LifeSciences).

Example 3—Competitive FACS with PD-L1 Antibodies

Chinese hamster ovary (CHO) cells were transfected with a PD-L1expression vector and subsequently selected for expression of theprotein (PD-L+ cells). The CHO cells were incubated with 1 μg/mlbiotin-labeled PD-1 for 1 hour.

Following incubation with biotin-labeled PD-1, anti-PD-L1 antibodieswere added to the supernatant at 4-fold dilutions, starting at 10 μm/ml,and allowed to incubate for 1 hour. The cells were washed and thencontacted with streptavidin-PE. Streptavidin-PE staining was analyzed byflow cytometry to determine percent inhibition of PD-1 binding by theanti-PD-L1 antibodies.

TABLE 3 Clinical Control mAb CTI-09 CTI-48 CTI-50 CTI-58 IC₅₀, g/ml9.19e−007 4.156e−005 9.985e−007 1.037e−006 3.463e−006

IC₅₀ values for several antibodies, including Clinical Control mAb (asdefined by the VH domain represented by SEQ ID NO: 73 and the VL domainrepresented by SEQ ID NO: 74), CTI-09, CTI-48, CTI-50, and CTI-58 werecalculated and can be found in Table 3. FIG. 1 shows the results of thisstudy.

Example 4—Antibody Binding Kinetics, Specificity, and Selectivity

Octet data analysis was used in determining affinity measurements toassess antibody binding kinetics. 2 mL of the loading sample wasprepared at 20 ug/mL (default concentration) in kinetic buffer. Aliquotat least 200 uL into a black 96-well plate. Concentration ranges for thesample were based on the estimated K_(D) of the interaction (ifavailable). Generally, the serial dilution was in a range from 0.1 K_(D)to 10 K_(D). A 7-point dilution was made into the sample column usingkinetic buffer as the sample diluent. The last well of the sample columnwas used as a reference well later in data analysis, should only containkinetic buffer.

Biosensors were hydrated in kinetic buffer (1×PBS, 0.1% BSA, 0.02%Tween20, 0.05% Sodium Azide) at room temperature for 10 minutes.

TABLE 4 Sample Loading ID Sample ID KD (M) kon(1/Ms) kdis (1/s) huPDL1CTI-48 8.47E−10 7.20E+05 6.10E−04 msPDL1 CTI-48 N/A N/A N/A cynoPDL1CTI-48 5.55E−10 1.14E+06 6.35E−04

Kinetic values for one exemplary anti-PD-L1 antibody, CTI-48, can befound in Table 4, and experimental results are shown in FIG. 2.

Example 5—Antibody Binding Affinity

ForteBio affinity measurements were performed generally as previouslydescribed (see, e.g., Estep et al., 2013). Briefly, ForteBio affinitymeasurements were performed by loading IgGs on-line onto AHQ sensors.Sensors were equilibrated off-line in assay buffer for 30 min and thenmonitored on-line for 60 seconds for baseline establishment. Sensorswith loaded IgGs were exposed to 100 nM antigen for 5 minutes,afterwards they were transferred to assay buffer for 5 min for off-ratemeasurement. Kinetics were analyzed using the 1:1 binding model.

TABLE 5 VH CDR3 IgG K_(D) (M) Ab Name Lineage monovalent kon (1/Ms) koff(1/s) CTI-09 1 N.B. CTI-48 1 8.24E−10 7.68E+05 6.33E−04 CTI-49 12.31E−09 7.28E+05 1.68E−03 CTI-76 1 8.24E−09 6.62E+05 5.45E−03 CTI-77 13.25E−09 5.44E+05 1.77E−03 CTI-78 1 3.46E−09 6.18E+05 2.14E−03 CTI-50 11.91E−09 7.94E+05 1.52E−03 CTI-07 2 7.97E−08 4.92E+05 3.92E−02 CTI-58 22.41E−08 4.61E+05 1.11E−02 Clinical Control NA 9.5E−10 mAb CTI-57 2 8.6E−10  5.2E+05  4.5E−04 CTI-97 1 1.82E−09 5.11E+05 9.28E−04 CTI-98 11.70E−09 5.02E+05 8.52E−04

Binding values for several exemplary antibodies are shown in Table 5.

Example 6—ADCC Activity of Anti-PD-L1 Antibodies

Reporter bioassays were performed in order to determineantibody-dependent cell-mediates cytotoxicity (ADCC) of the disclosedanti-PD-L1 antibodies. The assays utilized SUDHL-1 lymphoma cells anddonor PBMCs. Various antibodies were tested at concentrations of 1 or 3ug/ml.

The results of this study, which are shown for exemplary anti-PD-L1antibody CTI-48 in FIG. 3, are represented by percent cytotoxicityfollowing a 4 hour incubation with the disclosed antibodies.

Example 7—Immunoblockade Reporter Assay

Immunoblockade assays were performed using a PD1/PD-L1 Blockade AssayKit (Promega, CS187111) in 96 well plates. Three major events occur inthe assay. Event 1: TCR-mediated NFAT activation occurs when engineeredJurkat PD-1 Effector cells and aAPC (artificial antigen presenting cell)PD-L1 cells are engaged through TCR/TCR activator interaction. Event 2:Inhibition of NFAT signal by PD-1:PD-L1 ligation when no blockingantibodies are present. Event 3: Recovery of NFAT signal by addition ofanti-PD-1 or anti-PD-L1 blocking antibody.

The day before assay, 25 mL of cell recovery medium (10% FBS/F-12) wasmade in 50 mL conical tubes for Thaw-and-Use PD-L1 cells by adding 2.5mL FBS to 22.5 mL F-12. One vial of Thaw-and-Use PD-L1 cells (CS187103)was removed from freezer storage and transferred to the bench on dryice. The viral was thawed in a 37° C. water bath until cells are justthawed (about 3-4 minutes). The cell suspension was gently mixed in thevial by pipetting up and down, and transfer all the cells (0.5 mL) tothe tube labeled “PD-L1 cells” containing 14.5 mL cell recovery mediumfollowed by gentle inversions. The cell suspension was transferred to asterile reagent reservoir. Immediately, using a multichannel pipette,and 100 μL of cell recovery medium was added per well to outside wellsfor assay plates. The plates were incubated overnight in a CO2 incubatorat 37° C.

On the day of assay, fresh assay buffer (RPMI 1640+1% FBS) was prepared,and seven-point three-fold serial dilutions were made in assay bufferfor each of the test antibodies at 2× of final concentration. 95 μL ofmedium was removed from all the wells on the assay plates, and 40 μL ofserial dilutions of the test antibodies was added to the wellscontaining PD-L1 cells. 80 μL per well assay buffer was added to theoutside wells for each plate.

Thaw-and-Use PD-1 Effector cells (CS187105) were transferred into theassay plates, and the plates were incubated for six hours at 37° C. inCO2 incubator. After the six-hour induction, the assay plates wereremoved from the CO2 incubator and equilibrated at ambient temperaturefor 5-10 min. 80 μL of Bio-Glo™ Reagent was added to every test well,and the plates were incubated for another 5-10 min at ambienttemperature. Luminescence was measured in POLARstar Omega plate readerwith 0.5 sec integration.

The following antibodies were tested with final concentrations of 10μg/mL, 3.33 μg/mL, 1.11 μg/mL, 0.37 μg/mL, 0.123 μg/mL, 0.041 μg/mL, and0.014 μg/mL: CTI-2, Clinical Control mAb, CTI-09, CTI-48, CTI-50,CTI-07, and CTI-58.

Results for exemplary antibodies including Clinical Control mAb, CTI-48,and CTI-49 are shown in Table 6 below.

TABLE 6 Clinical Control mAb CTI-48 CTI-49 EC₅₀, g/mL 9.213e−0087.750e−008 9.191e−008

Example 8—PD-L1/B7.1 Inhibitor Screening Assay

A commercially available assay kit was used to screen and profile theinteraction of the disclosed antibodies and the PD-L1/B7.1 interaction.The kit came in a 96-well format with biotin-labeled B7-1 (CD80),purified PD-L1, streptavidin-labeled HRP, and assay buffer for 100binding reactions. The kit was used to detect biotin-labeled B7.1 bystreptavidin-HRP.

First, PD-L1 was coated on a 96-well plate. Next, either one of thedisclosed antibodies, a positive control, a substrate control, or ablank was added to each well and incubated prior to the addition ofB7.1-biotin. Finally, the plate is treated with streptavidin-HRPfollowed by addition of an HRP substrate to produce chemiluminescence,which can then be measured using a chemiluminescence reader.

The following antibodies were tested for their inhibitory effect on thebinding of PD-L1 and B7.1 at concentrations of 30 μg/mL, 10 μg/mL, 3.33μg/mL, 1.11 μg/mL, 0.37 μg/mL, and 0.123 μg/mL: CTI-1, CTI-2, CTI-33,CTI-48, and CTI-55.

Exemplary results indicate that the IC₅₀ for binding inhibition of thedisclosed antibodies ranges between 0.1816 and 0.5056 μg/mL. Forinstance, the IC₅₀ of CTI-48 was calculated to be 0.1816 μg/mL. Acomparison of the activity of CTI-48 and a clinical control mAb is shownin FIG. 5.

Example 9—Effect of Antibodies on IFN-γ Production

Antibodies were dosed into mixed lymphocyte reaction (MLR) cultures inorder to determine the effects of the disclosed antibodies on IFN-γproduction. The fold change in production of IFN-7 was determined aftera 4-day MLR culture with antibodies at a concentration of 10 μg/mL.Exemplary results, including those of an appropriate isotype control(hIgG1). As shown in FIG. 6, CTI-48 induced a comparable response to aclinical control mAb and many of the tested antibody elicited astatistically significant increase in IFN-γ production, including aroughly 10-fold increase by CTI-33 and CTI-55 over control levels.

All patents and publications mentioned in the specification areindicative of the levels of those of ordinary skill in the art to whichthe disclosure pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

Further, one skilled in the art readily appreciates that the presentdisclosure is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein.Modifications therein and other uses will occur to those skilled in theart. These modifications are encompassed within the spirit of thedisclosure and are defined by the scope of the claims, which set forthnon-limiting embodiments of the disclosure.

What is claimed:
 1. An antibody or a functional fragment thereof thatbinds to human programmed death-ligand 1 (PD-L1), wherein the antibodyor functional fragment thereof comprises: a heavy chain comprising:CDRH1 comprising amino acids 1-3 and 7-9 of SEQ ID NO: 1, wherein aminoacids 4-6 are not SSY; CDRH2 comprising amino acids 9-17 of SEQ ID NO:8; and CDRH3 comprising SEQ ID NO: 15; and a light chain comprisingCDRL1 comprising SEQ ID NO: 20; CDRL2 comprising SEQ ID NO: 21; andCDRL3 comprising SEQ ID NO: 22, SEQ ID NO: 75, or SEQ ID NO:
 76. 2. Theantibody or functional fragment thereof according to claim 1, whereinthe CDRH1 region comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 2, 3, 4, 5, 6 and
 7. 3. The antibody orfunctional fragment thereof according to claim 1, wherein the CDRH2region comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 8, 9, 10, 11, 12, 13 and
 14. 4. The antibodyor functional fragment thereof according to claim 1, wherein thevariable heavy framework region 1 comprises SEQ ID NO.
 26. 5. Theantibody or functional fragment thereof according to claim 1, whereinthe variable heavy framework region 1 comprises SEQ ID NO.
 27. 6. Theantibody or functional fragment thereof according to claim 1, whereinthe variable heavy framework region 2 comprises SEQ ID NO.
 28. 7. Theantibody or functional fragment thereof according to claim 1, whereinthe variable heavy framework region 3 comprises SEQ ID NO.
 29. 8. Theantibody or functional fragment thereof according to claim 1, whereinthe variable heavy framework region 4 comprises SEQ ID NO.
 30. 9. Theantibody or functional fragment thereof according to claim 1, whereinthe variable light framework region 1 comprises SEQ ID NO.
 31. 10. Theantibody or functional fragment thereof according to claim 1, whereinthe variable light framework region 2 comprises SEQ ID NO.
 32. 11. Theantibody or functional fragment thereof according to claim 1, whereinthe variable light framework region 3 comprises SEQ ID NO. 33, SEQ IDNO. 77, or SEQ ID NO.
 78. 12. The antibody or functional fragmentthereof according to claim 1, wherein the variable light frameworkregion 4 comprises SEQ ID NO.
 34. 13. The antibody or functionalfragment thereof according to claim 1, wherein the variable heavy chainsequence comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOS: 36, 37, 38, 39, 40 and
 41. 14. The antibody orfunctional fragment thereof according to claim 1, wherein the variableheavy chain sequence comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 51, 52, 53, 54, 55 and
 56. 15. Theantibody or functional fragment thereof according to claim 1, whereinthe variable light chain sequence comprises SEQ ID NO:
 42. 16. Apharmaceutical composition comprising the antibody or functionalfragment thereof according to claim
 1. 17. An antibody or a functionalfragment thereof that binds to human programmed death-ligand 1 (PD-L1),wherein the antibody or functional fragment thereof comprises a heavychain comprising SEQ ID NO:
 35. 18. A method of inhibiting cancer cellproliferation in a subject, comprising administering an antibody orfunctional fragment that binds human PD-L1 with a K_(D) of at least8.30×10⁻⁹ and CDRH3 comprising SEQ ID NO: 15 to a subject with cancer.19. The method of claim 18, wherein the cancer is a hematologicalcancer, a neurological cancer, breast cancer, a gastrointestinal cancer,renal cell carcinoma, or a genitourinary cancer.
 20. The method of claim18, wherein the method further comprises a step of administering to thesubject an additional therapeutic compound.
 21. The method of claim 20,wherein the additional therapeutic compound is a CAR-T cell, atumor-targeting antibody, an immune response potentiating modality, or asmall molecule drug.
 22. A method of inhibiting tumor cell proliferationin a subject, comprising administering an antibody or functionalfragment thereof that binds human PD-L1 with a K_(D) of at least2.50×10⁻⁸ and CDRH3 comprising amino acids 6-13 of SEQ ID NO: 18 to asubject with cancer.
 23. The method of claim 22, wherein the methodfurther comprises a step of administering to the subject an additionaltherapeutic compound.
 24. The method of claim 23, wherein the additionaltherapeutic compound is a CAR-T cell, a tumor-targeting antibody, animmune response potentiating modality, or a small molecule drug.